Molecular Identification of Bupleurum chinense Seeds Based on DNA Barcoding Technology / 中国实验方剂学杂志

Objective:To establish a molecular identification method for Bupleurum chinense seeds based on ribosomal DNA internal transcribed spacer (ITS) sequence, ensuring the species authenticity of the cultivated seeds of B. chinense. Method:A total of 59 seeds samples of B. chinense and its main cultivated species, marketed B. chinense were collected. The effect of different sampling amounts and different water bath conditions on DNA extraction quality of the seeds was investigated, a DNA extraction method for seeds of Bupleurum was established. Their ITS sequences were obtained by polymerase chain reaction (PCR) and bidirectional sequencing. In addition, 34 ITS sequences of main cultivated Bupleurum species, such as B. chinense, B. scorzonerifolium, B. falcatum and B. smithii, were downloaded from GenBank to enrich identification database of B. chinense seeds. The neighbor-joining (NJ) dendrogram were constructed by MEGA-X 10.0.5 software to investigate the the species identification ability of ITS sequences for B. chinense seeds. And DNA barcoding identification of marketed B. chinense seeds was conducted based on BLAST method and NJ dendrogram method. Result:In total, 59 ITS sequences were obtained. ITS sequences of B. chinense could be divided into six haplotypes, including seven variable sites. The NJ dendrogram indicated that all the haplotypes of B. chinense could form independent branches, which could be distinguished from other cultivated species of Bupleurum in the collected samples, and possessed the ability to identify species of B. chinense seeds. Based on ITS sequence barcoding identification, 3 of the 19 marketed B. chinense seeds were B. falcatum with a counterfeit rate of 15.8%. Conclusion:DNA barcoding technology based on ITS sequence can accurately and reliably identify B. chinense seeds and its adulterants, providing reference for the standardization construction of Chinese medicinal materials seeds.

Isolation and identification of endophytic fungi from Huperzia serrata and their metabolites' inhibitory activities against acetylcholinesterase and anti-inflammatory activities / 中国中药杂志

A total of 27 endophytic fungal strains were isolated from Huperzia serrata,which were richly distributed in the stems and leaves while less distributed in roots. The 27 strains were identified by Internal Transcribed Spacer( ITS) r DNA molecular method and one of the strains belongs to Basidiomycota phylum,and other 26 stains belong to 26 species,9 general,6 families,5 orders,3 classes of Ascomycota Phylum. The dominant strains were Colletotrichum genus,belonging to Glomerellaceae family,Glomerellales order,Sordariomycetes class,Ascomycota Phylum,with the percentage of 48. 15%. The inhibitory activities of the crude extracts of 27 endophytic fungal strains against acetylcholinesterase( ACh E) and nitric oxide( NO) production were evaluated by Ellman's method and Griess method,respectively. Crude extracts of four fungi exhibited inhibitory activities against ACh E with an IC50 value of 42. 5-62. 4 mg·L~(-1),and some fungi's crude extracts were found to inhibit nitric oxide( NO) production in lipopolysaccharide( LPS)-activated RAW264. 7 macrophage cells with an IC50 value of 2. 2-51. 3 mg·L~(-1),which indicated that these fungi had potential anti-inflammatory activities.The chemical composition of the Et OAc extract of endophytic fungus HS21 was also analyzed by LCMS-IT-TOF. Seventeen compounds including six polyketides,four diphenyl ether derivatives and seven meroterpenoids were putatively identified.

DNA barcoding sequence analysis of Amomum tsao-ko germplasm resources in Yunnan Province / 中草药

Objective: To screen and evaluate DNA barcoding of Amomun tsao-ko populations in Yunnan. Methods: ITS, psbA-trnH, matK, rbcL, and ycf1 sequences were screened and evaluated using A. tsao-ko as samples. The samples of A. tsao-ko population were amplified and sequenced. The sequences were spliced with Genestar, and then processed with Mega for data processing. And A. tsao-ko diversity and identification were analyzed and discussed. Results: The length of the amplified fragments of primers ITS5 and ITS4 was approximately 520 bp; The length of the amplified fragments of the primers rbcLa-F and rbcLa-R was approximately 498 bp; The length of the amplified fragments of the primers ycf1-bF and ycf1-bR was approximately 800 bp; The length of the amplified fragments of the primers psbA-trnH-1F and psbA-trnH-1R was approximately 400 bp; The length of the amplified fragments of the primers matK-2F and matK-2R was approximately 470 bp. The success rate of amplification and sequencing was high, and most of the results were available. By analyzing the amplification results of ITS, psbA-trnH, matK and ycf1 sequences of A. tsao-ko, A. tsao-ko and other Amomum genus plants can be clearly distinguished; All samples of the ITS sequence were divided into MG5 white flower A. tsao-ko population and other populations; All samples of the psbA-trnH sequence were divided into MG5 white flower A. tsao-ko population, MG6 yellow flower A. tsao-ko population and other populations; All samples of the matK sequence were divided into MG6 A. tsao-ko population and other populations. The MG5 white flower A. tsao-ko sample failed to be amplified; All samples of the ycf1 sequence were divided into the MG6 yellow flower A. tsao-ko population and other populations, and the MG5 white flower A. tsao-ko population was clustered with the other 22 A. tsao-ko populations; The amplification of rbcL sequence was consistent for all samples. Conclusion: The ITS, matK, psbA-trnH and ycf1 sequences can accurately distinguish A. tsao-ko from other plants of Amomum genus; The sequence site variations were found in matK, psbA-trnH and ycf1 sequences of MG6. This research has contributed to the selection and breeding of A. tsao-ko varieties. ITS and psbA-trnHsequences can distinguish yellow flower and white flower of A. tsao-ko; There is no variation in the rbcL sequence of all samples of white and yellow flowers of A. tsao-ko, and Amomum tsao-ko and other plants of Amomum genus cannot be identified with the rbcL sequence, which can be discarded.

Cloning and sequence analysis of rdna its from plants in genus asarum l / 中草药

Objective To clone and analyze ITS sequences of eight Asarum L. medical plants, and to provide evidence for both molecular identification and genetic diversity studies. Methods PCR amplifications were performed with genomic DNA as templates to isolate ITS sequences, and sequence comparisons as well as phylogenetic tree construction were conducted by bioinformatics software. Results Sequencing results indicated that the full ITS sequences of eight Asarum plants ranged from 637 to 646 bp in length, and 5.8 S sequence was highly conserved with identical sequence length and nucleotide composition. However, sequence variations were observed in ITS1 and ITS2, their sequences were 255-257 bp and 226-232 bp in length, respectively. Insertion/deletion and conversion/transition events were occurred frequently in ITS1 and ITS2 sequences, with variable sites of 46 and 30 and parsimony informative sites of 14 and 9, respectively. Results of the phylogenetic analysis indicated that the eight plants grouped to four different clades, which were completely coincide with traditional plant taxonomy. Groups of I-IV corresponded to section Asarum L., Asiasarum L., Longiflora L., and Heterotropa (Nakai) Hara, respectively. Conclusion ITS sequences of eight Asarum plants were rich in informative sites, which could be applied as molecular marker to identify these species.

Identification and characterization of a taxol-producing endophytic fungus from Taxus media / 生物工程学报

To enrich the resource pool of endophytic fungi from plants which produce taxol, a taxol-producing endophytic fungus TMS-26 was isolated from the stem of Taxus Media. The result of high performance liquid chromatography (HPLC) showed that TMS-26 extract exhibited similar chromatographic peaks and retention time (4.545 min) with authentic taxol. Then mass spectrometry (MS) analysis further confirmed that TMS-26 extracts contained the same mass peaks with authentic taxol ((M+Na)+=876). These indicated that the isolated endophytic fungus TMS-26 can produce taxol. According to the morphological characteristics, the molecular analysis of 18S rDNA and internal transcribed spacer nuclear rDNA gene sequence, the fungus was identified as Aspergillus fumigatus TMS-26.

rDNA-ITS Sequence analysis of traditional Mongolian medicinal plants in Vicia L / 中草药

Objective: To find the patterns of the rDNA ITS sequence variation of eight traditional Mongolian medicinal plants in Vicia L. (including V. amoena, V. ramuliflora, V. cracca, V. multicaulis, V. amurensis, V. japonica, V. pseudorobus, and V. unijuga), and establish the molecular biological method for the identification of the eight plants. Methods: DNA was extracted from the dry leaves, and the ITS gene fragments were PCR amplified and sequenced. The sequence features were compared and analyzed using Clustal X and MEGA softwares. Results: The length of ITS region varied from 598-611 bp with 40 variable sites, 33 parsimony information sites, and 29 singleton sites among eight traditional mongolian medicinal plants in Vicia L. Sequences were submitted to NCBI database with the registry numbers of JQ309787-JQ309794, KJ417905-KJ417931. And ITS sequences of five species among them were first reported in the world, they were V. amurensis (JQ309789), V. japonica (JQ309792), V. multicaulis (JQ309791), V. pseudorobus (JQ309788), and V. ramuliflora (JQ309793). Conclusion: ITS sequences could be used to authenticate the eight species in Vicia L. and also could provide a reference for molecular identification.

Phylogenetic Diversity and Antifungal Activity of Endophytic Fungi Associated with Tephrosia purpurea

Sixty-one endophytic fungus strains with different colony morphologies were isolated from the leaves, stems and roots of Tephrosia purpurea with colonization rates of 66.95%, 37.50%, and 26.92%, respectively. Based on internal transcribed spacer sequence analysis, 61 isolates were classified into 16 genera belonging to 3 classes under the phylum Ascomycota. Of the 61 isolates, 6 (9.84%) exhibited antifungal activity against one or more indicator plant pathogenic fungi according to the dual culture test. Isolate TPL25 had the broadest antifungal spectrum of activity, and isolate TPL35 was active against 5 plant pathogenic fungi. Furthermore, culture filtrates of TPL25 and TPL35 exhibited greater than 80% growth inhibition against Sclerotinia sclerotiorum. We conclude that the endophytic fungal strains TPL25 and TPL35 are promising sources of bioactive compounds.

Sequences analysis and pattern recognition of ITS in Sarcandra glabra from different areas / 中草药

Objective: To study the ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences of Sarcandra glabra from different areas and five plants in Chloranthus Swartz, and to provide pattern recognition and thread for the species identification of S. glabra. Methods: The ITS sequences of 18 populations of S. glabra and six populations of Chloranthus spicatus were amplified by PCR with universal primer of ITS and sequenced, and the ITS sequences of the other plants in Chloranthus Swartz were searched in GeneBank. Data were analyzed by ClustalX 2.1, and a cluster analysis was presented by UPGMA. Results: The semblance of ITS sequences of S. glabra from different areas was 99%. The total mutation rate of ITS1 sequence (2.7%) was higher than that of ITS2 sequence (1.4%). However, compared with other plants in Chloranthus Swartz, the total mutation rate of ITS2 sequence (20.3%-22.7%) was higher than that of ITS1 sequence (15.9%-18.3%). The cluster analysis showed that there was little variation among the 18 populations of S. glabra, but there was significant difference with the five plants of Chloranthus Swartz. Conclusion: ITS sequence can be used to identify the plants from different areas and in different genus, ITS sequence of S. glabra has several specified information sites to identify the five plants in Chloranthus Swartz, with significantly different cluster analyseis, and is the active molecular marker for the species identification of S. glabra and plants in Chloranthus Swartz.

Sequence analysis on rDNA-ITS region of germplasm resources from Dioscoreae Rhizoma / 中草药

Objective: To provide the scientific evidence for molecular identification and phylogenetic evolution by analyzing internal transcribed spacer (ITS) sequences in 14 different germplasms from Dioscoreae Rhizoma. Methods: The ITS regions were cloned by PCR amplification and sequenced bi-directionally. The ITS sequences were aligned using Clustal X software (version 1.83), the genetic distances were calculated using Mega software (version 4.1), and the phylogenetic trees were constructed through the Neighbor-joining (NJ) and maximum parsimony (MP) methods. Results: The length of ITS sequences obtained ranged from 558 to 594 bp, of which ITS1 was from 141 to 165 bp and ITS2 was from 146 to 158 bp. There were a lot of transitions and transversions in ITS sequences, and the transition/transversion ratio among 14 different germplasms was 5.347. Both in ITS1 and ITS2, 102 variable sites were further observed. The kimura 2-parameter (K2-P) genetic distance among 14 different germplasms ranged form 0 to 0.5172. The phylogenetic trees suggested that there were close genetic relationship among Dioscorea opposita, D. persimilis, and D. japonica, they grouped closely into Clade I; Furthermore, D. alata and D. fordii also shared a closer genetic relationship comprised another clade (Clade II). Conclusion: The phylogenetic analyses based on ITS sequences can present a foundation for clearing the evolution of germplasm resources in Dioscoreae Rhizoma; The variable bases can also provide reliable molecular evidences for identifying different genotypic germplasms of Dioscoreae Rhizoma.

Identification of 17 species of Angelicae Pubescentis Radix based on ITS analysis / 中草药

Objective: To provide the evidence for the molecular identification of Angelicae Pubescentis Radix (APR) and discuss the scientific classification standard through ITS analysis on 26 samples of APR from 17 species. Methods: ITS of 26 populations was amplified and sequenced. The differences among the different samples were compared and K2P genetic distances of ITS sequence were calculated. NJ tree was constructed and haplotype network map was obtained by using the Network 4.2.0.1 software. Results: NJ tree and haplotype network map suggested that 26 populations were clustered into five groups, and the 17 APR could be sorted into four types after comprehensive analysis. Angelica pubescens was different from other species due to the distinct base sequence in the ITS sequence. All species were identified by ITS analysis except for the three species in Heracleum L. Conclusion: The ITS sequence is powerful for the identification and classification of medicinal APR. Tetrataenium-type plant could be the first choice as succedaneum and followed by Heracleum-type plant, then Aralia-type plant be the last candidate as well.

Isolation and identification for strains from wild Phellinus and analysis on its secondary metabolites / 中草药

Objective: To isolate and identify the strains from wild Phellinus linteus and analyze its secondary metabolites. Methods: Strains isolated and purified were investigated by optical microscope to observe the characteristics of mycelia, ITS sequence was used for molecular identification and its secondary metabolite content was analyzed by chemical colorimetry. Results: The morphological characteristics of mycelia from pinus, Morus alba, Syringa reticulata, and populus were the same. The growth rate of four flamentous colonies and their colors were different, wherein the growth rate of the colony from M. alba was the fastest, up to 0.47 cm/d. Four strains all belonged to genus Phellinus sp, where strain from pinus was identified as P. pini, M. alba was P. linteus, S. reticulata and populus were P. baumii. The species of the secondary metabolites in the sporocarp and mycelia of Phellinus were the same, but the content was different. For the sporocarp, the highest polysaccharide content was in the sporocarp from S. reticulata with the content of 98.20 mg/g, the highest flavonoids, terpenoids, and polyphenols contents were all in the sporocarp from poplar with the contents of 548.49, 1.48, and 33.70 mg/g, respectively. For mycelia, the highest polysaccharide, flavonoids, terpenoids, and polyphenols contents were all in the mycelia of M. alba, the contents were 259.64, 223.11, 43.78, and 24.80 mg/L, respectively. Additionally, the content of terpenoids in the mycelia was higher than that of the sporocarp. Among them, the content of terpenoids in the mycelia from M. alba was about 7 times as that in fruit body. Conclusion: There are no differerences amony form of mycelia from four strains of P. linteus. rDNA ITS sequence analysis could be used for the identification of strains. The species four Phellinus strains and its secondary metabolite content are clarified.

ITS sequences and phylogenetic relationships of dandelions from different areas / 中国药学杂志

OBJECTIVE: To study the genetic characteristics and phylogenetic relationships of dandelions of 20 populations by sequencing ITS sequence. METHODS: The ITS sequence was amplified by PCR with universal primer of ITS, and the ITS sequence of the PCR products was directly sequenced after purification. RESULTS: The total length of ITS sequence of different samples was 711 bp. The ITS sequence was divided into three fragments; ITS1 was 225 bp, 5.8S was 262 bp and ITS2 was 226 bp. In all samples 5.8S was comparatively conserved, while ITS1 and ITS2 had higher diversity in base sequence among the various specimens. CONCLUSION: ITS sequence can be used to analyze the phylogenetic relationships of different varieties of dandelions.

Field test and analysis for fungus inducing the formation of agilawood from Aquilaria sinensis / 中国药学杂志

OBJECTIVE: To investigate the feasibility of utilizing three fungi YNAS04, YNAS06 and YNAS08 to induce the formation of agilawood from Aquilaria sinensis in field and laboratory, respectively. METHODS: Agarwood was obtained by inoculating the fungus in the stem of A. sinensis under natural conditions, and the main ingredients of the volatile oil from induced agilawood were analyzed by GC-MS. The authenticity of the used fungi was detected by the method of morphology and ribosomal internal transcribed spacer. RESULTS: The weights of the induced materials from YNAS04, YNAS06 and YNAS08 were (1.786±0.473), (2964±0.492) and (3.615±0.591) g after six months, which were heavier than blank control (1.325±0.407) g and negative control (1.462±0.417) g, respectively. The fungus inoculated in the stem of A. sinensis and became the dominant fungi in the tissue under natural conditions. Sesquterpenoids and chromone analogues were detected from three fungi inducing materials of six months old. CONCLUSION: Utilizing fungi to induce the formation of agilawood from Aquilaria sinensis has the advantages of spending less time and producing high-quality secondary metabolites. This method has important application foreground in large-scale agilawood production.

ITS sequence analysis of eight medicinal plants in Clematis L / 中草药

Objective: To provide molecular evidence for identification of medicinal plants in Clematis L., sequencing and comparison of ITS from C. uncinata, C. henryi, C. chinensis, C. finetiana, C. lasiandra, C. patens, C. apiifolia, and C. patens ssp. tientaiensis were performed. Methods: ITS sequences were amplified from leaf genomic DNA by PCR. Sequence features were compared and analyzed using Clustal X, MEGA, and DNASTAR softwares. Results: Between ITS1 and ITS2 of the eight medicinal plants in Clematis L., the length of ITS region varied from 534-561 bp with 50 variable sites and 22 parsimony information sites. The smallest genetic distance was observed between C. patens ssp. tientaiensis and C. patens, and the largest existed between C. patens ssp. tientaiensis and C. apiifolia revealing their farest genetic relationship. Sequences were submitted to NCBI database with the registry numbers of JF714638-JF714645. Conclusion: ITS sequences of the eight medicinal plants in Clematis L. are obtained, which could provide a foundation for molecular identification.

Taxonomic Study on the Lichen Genus Coccocarpia (Lecanorales, Ascomycota) in South Korea

Three species of Coccocarpia have been reported from Korean Peninsular. However, there was no revisional study on this genus before. After careful examination of the specimens deposited in the Korean Lichen Research Institute (KoLRI) and collected from main mountain areas of Korea, two species of Coccocarpia, C. palmicola and C. erythroxyli, have been revealed to occur and confirmed in South Korea. The presence and absence of isidia and apothecia are the most important characters for the South Korean species. We provide the detailed description and illustration of the available two species. A key to the species is also provided.

Diversity of Endophytic Fungi Associated with Taraxacum coreanum and Their Antifungal Activity

Endophytic fungi were isolated from healthy leaf and root samples of Taraxacum coreanum. Of the 72 isolates recovered, 39 were from leaves and 33 from roots with an isolation frequency of 54% and 46%, respectively. Based on ITS sequence analysis, 72 isolates were classified into 19 genera of which 17 were under the phylum Ascomycota and 2 were under Basidiomycota. Diverse genera were found and Alternaria, Cladosporium, Fusarium and Phoma were dominant. Out of 19 genera, Apodus, Ceriporia, Dothideales, Leptodontidium, Nemania, Neoplaconema, Phaeosphaeria, Plectosphaerella and Terfezia were new to Korea. Seventy two isolates were screened for antifungal activity, of which 10 isolates (14%) were found active at least against one of the tested fungi. Isolate 050603 had the widest antifungal spectra of activity, and isolates 050592 and 050611 were active against three plant pathogenic fungi.

Analysis of rDNA ITS sequences in root tuber of Polygonum multiflorum from various habitats / 中草药

Objective To study the genetic diversity of rDNA ITS sequences in the root tuber of Polygonum multiflorum from various habitats and to analyze the utility of ITS sequences in molecular authenticating the genuineness, identifying the varieties of wild resources, and studying the germplasm resources. Methods Firstly, total DNA in the root tuber of P. multiflorum from various habitats was extracted. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and sequenced after purification. Results The total length of ITS sequence is 648 bp in the different samples, such as 5.8S, 18S, and 26S rDNA. The lengths of three fragments, ITS1, 5.8S, and ITS2, are 195 bp, 164 bp, and 189 bp, respectively. There are seventeen variable sites among the sequences. Conclusion ITS Sequence can be used to authenticate the genuineness and identify the varieties of wild resources in the root tuber of P. multiflorum.

ITS sequence analysis on medicinal plants of Euphorbia L. in Anhui and Jiangsu Provinces / 中草药

Objective To study the correlation and variation between plants of internal transcribed spacers (ITS) sequence of the six medicinal plants of Euphorbia L. in Anhui and Jiangsu Provinces, in (order) to provide their DNA molecular marker to identify and explore the phylogenetic relationship of the plants of [WTBX]Euphorbia L. Methods To determine rDNA ITS sequence for the plants of Euphorbia L. by PCR technology. Results The sequences of ITS1 in the six species ranged from 255 to 262 bp in length and those of ITS2 from 214 to 236 bp. The dendrogram was obtained with Mega2 analysis. The analysis result was consistent with those from morphology. Conclusion The method can be used to identify the plants of [WTBX](Euphorbia) L. among different species and to differentiate their fakes.

THE PHYLOGENETIC RELATIONSHIPS OF GRIFOLA UMBELLATA AND ITS COMPANION FUNGUS: EVIDENCE FROM ITS SEQUENCE ANALYSIS / 微生物学通报

The sequences of 5.8S rDNA and the flanking internal transcribed spacers (ITS1 and ITS2) were sequenced from hypha, fruit body and sclerotia of Grifola umbellata and its companion fungus. Their ITS sequences similarity was 99.36%. The results suggested that G. umbellata was closely related to its companion fungus.

Phylogenetic relationships of Dendrobium candidum and its related species based on DNA ITS sequences / 中成药

AIM: To apply molecular systematic techniques to revealing the genetic diversity of medicinal plant of Dendrobium candidum and its related species in Dendrobium.METHODS: The internal transcribed spacer(ITS) as well as 5.8S rDNA sequences of 40 samples of 25 species of Dendrobium were carried out on 25 dendrobi-um samples and amplified using PCR method and sequenced.Mega 3.1 was used to analyze the genetic diversity within the genus.Phylogenetic relationships among species in Dendrobium were estimated by maximum parsimony and neighbor-joining methods to construct similar ITS trees.RESULTS: The phylogenetic analysis indicated that Dendrobium candidum had a markedly difference in the interspecies and possessed the characteristic sequence site.CONCLUSION: ITS sequences as a good molecular marker for authentication between Dendrobium and outgroup is feasibility.